Affinity purification of BHC. (a) Purification scheme. HeLa nuclear extract (NE) was fractionated by using P11 chromatography. The 0.5 M KCl elution was concentrated on a DEAE-Sephacel column and purified further by using an anti-BRAF35 antibody column (α-BRAF35). The bound proteins were washed with buffer containing 0.5 M KCl and 0.2 M guanidine hydrochloride and eluted by using 0.2 M glycine, pH 2.5. (b) Polypeptide composition of the affinity-purified BHC. Affinity-purified BHC was analyzed by SDS/PAGE followed by silver staining or Colloidal blue. α-IgG represents the control preimmune IgG eluate. Molecular mass markers are shown to the left of the figure. (c) Primary amino acid sequence of BHC80. Peptides sequenced by ion-trap mass spectrometry are double-underlined. The acidic Q/P-rich region is underlined, and the leucine zipper and the PHD domains are represented by black and gray shadings, respectively. (d) Diagrammatic representation of BHC80 structural domains. (e) Reverse transcription–PCR representing the expression profile of BRAF35 and BHC80 in different human tissues. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Conventional purification of BHC. (a) Purification scheme. HeLa nuclear extract (NE) was fractionated by chromatography as described in Materials and Methods. The horizontal and diagonal lines indicate stepwise and gradient elution, respectively. Concentrations are given as molar. (b) Western blot analysis of the Mono S fractions (15 μl) using antibodies shown to the right of the figure. (c) Western blot analysis of immunoeluates of antibodies shown on the top of the figure using anti-BRAF35 antibodies. Input (I) is the BHC pool from the 0.5 M P11 fractions (10 μl). P11 (100 μl of 0.5 M) was used for immunoprecipitation.

Affinity purification of human BHC from Flag-BRAF35 cells. (a) Purification scheme. Nuclear extract (NE) from FBRAF35-4 cells was fractionated by using an anti-Flag M2 affinity column. Bound proteins were analyzed further by chromatography on a heparin-5PW column. (b) Silver-stain analysis of the anti-Flag affinity eluates (15 μl) corresponding to early (lane 1) and late (lane 2) eluting fractions. (c) Western blot, HDAC activity, and silver-stain analysis of the heparin-5PW column fractions (15 μl). Western blotting was performed by using antibodies shown to the right of the figure.

BHC mediates RE1-dependent transcriptional repression. (a) Graph of relative luciferase activity after cotransfection of 293 cells. Transfection and luciferase assay were carried out as described in Materials and Methods. The cells were cotransfected with the reporter plasmid of either type II promoter lacking the RE1 element (black) or RE1-containing type II promoter (gray) and various concentrations of BRAF35 (0.3, 1, or 2 μg of DNA) and BRAF35(K116I) (0.3, 1, or 2 μg of DNA) or with pFlag-CMV2 control vector (2 μg of DNA). The standard errors are indicated by the thin vertical lines. Each point represent six measurements, and the experiment was repeated six independent times. (b) Graph of relative luciferase activity after cotransfection of 293 cells with the reporter construct of RE1-containing type II sodium-channel promoter or the type II promoter lacking RE1 sites in addition to pFlag-CMV2 (2 μg of DNA), BHC80 (1 μg of DNA), BRAF35 (1 μg of DNA), HDAC1 (1 μg of DNA), and REST (1 μg of DNA). The standard errors are indicated by the thin vertical lines. Each point represents six measurements. (c) Graph of relative luciferase activity after cotransfection of 293 cells with the RE1-containg type II sodium-channel promoter and the following constructions: pFlag-CMV2 (2 μg of DNA), BHC80 (1 μg of DNA), SNF2h (1 μg of DNA), and REST (1 μg of DNA). The standard errors are indicated by the thin vertical lines.

BHC is recruited to the endogenous synapsin gene. (a) ChIP in 293 cells using antibodies shown on the top of the figure. PCR products corresponding to synapsin, PFC, and ELK1 promoters are displayed as 101-, 313-, and 120-bp bands on an ethidium bromide-stained agarose gel, respectively. (b) Western blot analysis using anti-Flag antibodies after transfection of Flag-BRAF35 or Flag-BRAF35(K116I) in 293 cells. (c) ChIP was performed by using formaldehyde-treated HeLa or 293 cells, respectively. After transfection with either Flag-BRAF35 or Flag-BRAF35(K116I), antibodies to REST, CoREST, HDAC2, and the Flag epitope were used to immunoprecipitate complexes associated with the synapsin RE1 sites. The PCR products are displayed as 101-, 113-, and 277-bp bands on an ethidium bromide-stained agarose gel. (d) Western blot analysis using antibodies shown to the left of the figure of Flag-affinity eluate purified using 293 cells stably expressing BRAF35(K116I). NE, nuclear extract.